CN112505320A - Ampicillin residue detection method and application - Google Patents
Ampicillin residue detection method and application Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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Abstract
The invention discloses a method for detecting ampicillin residues, which utilizes the characteristic that ampicillin can be separated from the surface of gold nanoparticles by utilizing the specific binding energy of ampicillin and aptamer, amplifies signals by utilizing hybrid chain unzipping (HCR), and establishes a high-sensitivity detection method for ampicillin residues through the change of an absorption spectrum of a gold nanoparticle solution. The HCR amplification nanogold colorimetric method of the ampicillin aptamer has the advantages of simplicity and convenience in operation, rapidness in melting, high sensitivity and the like, and can be used for quickly detecting the residual quantity of ampicillin.
Description
Technical Field
The invention relates to the technical field of ampicillin residue detection, in particular to a detection method and application of ampicillin residue.
Background
Ampicillin (AMP) is used as a broad-spectrum antibiotic, is a beta-lactam broad-spectrum antibiotic, has the function of promoting the growth of animals besides infection resistance, so that Ampicillin (AMP) is widely applied to the agricultural field such as livestock and poultry and aquaculture, is widely applied to the aspects of human life, brings great economic benefit to human beings, simultaneously abuses a large amount of antibiotics, causes antibiotic residues in animals and plants, and further brings threat to the nature and human beings through enrichment effect. Ampicillin residues have many adverse effects on human beings, such as anaphylaxis, respiratory disorder, epilepsy, etc. Along with the improvement of living standard of people, food safety is more and more emphasized by people, and the problem of antibiotic residue is more and more concerned by people.
At present, common detection methods for ampicillin, such as microbiological detection method, immunological detection method, chromatography combined technology and the like, are difficult to be put into large-scale use due to the defects of complex operation, high cost, strict requirement on a reaction system and the like. In addition, the nano gold is also called colloidal gold and is a spherical nano material with the particle size of 1-100 nm. The aptamer is a detection method appearing in recent years, and the method has the advantages of stable performance, simplicity in operation, wide target molecule range and the like. The nano gold has unique optical characteristics, the aqueous solution of small-particle-size gold nanoparticles (10-100 nm) is generally wine red, the absorption peak is generally near 520nm, when the nano gold is aggregated under the external action, the absorption peak is red-shifted to a region near 620nm, and the color of the nano gold is changed from wine red to blue-violet. An aptamer is an oligonucleotide biorecognition molecule that binds highly specifically to a target under test based on spatial complementarity.
Therefore, according to the above, how to provide a method for detecting ampicillin residue by using a nanogold aptamer HCR amplified colorimetry is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a method for detecting ampicillin residues, which is a method for efficiently detecting ampicillin by designing and developing a hairpin DNA probe, using an aptamer as an initiation chain, and initiating a complementary effect of the aptamer and the hairpin DNA probe through the combination of the aptamer and ampicillin to further initiate a cascade cross-complementary amplification effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection method of ampicillin residues comprises the steps of firstly designing an ampicillin aptamer sequence and a matched hairpin DNA probe, preparing an HCR solution, and determining the ampicillin residues through the HCR solution and the nanogold solution by using an HCR amplified nanogold colorimetric method;
wherein the sequence of the ampicillin aptamer is as follows:
AMP-1：5’-CCGCCCGCTTAGTAGTTACGCTTACCTCTTGTTAGCGGG CGGTTGTATAGCGG-3’；SEQ ID NO.1；
the sequence of the hairpin DNA probe was:
H1：5’-CATCTCGGTTTGGCTTTCTTGTTACGGGCATTAGTAGTTACG CTTACC-3’；SEQ ID NO.2；
H2：5-TAACAAGAAAGCCAAACCGAGATGGGTAAGCGTAACTAGT AATGCCCG-3’；SEQ ID NO.3。
preferably, the method specifically comprises the following steps:
s1, synthesis of ampicillin aptamer and matched hairpin DNA probe: designing the sequence of the ampicillin aptamer as follows: AMP-1, with the sequence of the hairpin DNA probe: h1 and H2, all of which are synthesized by Shanghai Bioengineering, Inc.;
s2, preparation of HCR solution: the H1, H2 and AMP-1 nucleic acid sequences in step S1 were used with ddH2O preparing 1 mu M of nucleic acid working solution respectively, andmelting at 90-95 deg.c for 1-20min, slowly cooling to 4-30 deg.c within 0.5-2 hr, and mixing the treated AMP-1 aptamer with the treated H1 and H2 hairpin nucleic acid probe to obtain HCR solution stored at-80 deg.c to-20 deg.c;
s3, preparing a nano gold solution: preparing a clear and transparent 0.01-0.1% chloroauric acid solution from chloroauric acid, continuously heating to boil under stirring, adding a 0.1-5% trisodium citrate solution, continuously boiling and stirring, changing the solution from light gray to blue and purple in a beaker to finally obtain wine red, namely a nanogold solution, keeping stirring until the solution is cooled to room temperature, subpackaging and storing at 4-30 ℃, and representing the aggregation state of the nanogold solution by using a transmission electron microscope;
s4, detection of ampicillin residues: respectively taking the nanogold solution and the HCR solution, adding ampicillin with different concentrations, incubating for 0-120min at room temperature, adding 0.1-4M NaCl solution, incubating at room temperature, detecting absorbance by an enzyme-linked immunosorbent assay, and analyzing to obtain a linear equation; and testing the absorbance of the food to be tested by adopting the same method, substituting the absorbance into a linear equation, and obtaining the residual amount of ampicillin in the food.
Preferably, in step S2, the volume ratio of the treated AMP-1 aptamer, the treated H1 and the H2 hairpin nucleic acid probe is: (1): (1-10): (1-10).
Preferably, in step S3, the volume ratio of the chloroauric acid solution to the trisodium citrate solution is: (100-2000): (1-5).
Preferably, in step S4, the volume ratio of the nano-gold solution, the HCR solution and the sodium chloride solution is: (1): (1-10): (1-10)(100-1000): (10-100): (100-1000).
Preferably, the linear equation is y-0.1636 x +0.025, R2The detection range for ampicillin was 0-1.2. mu. mol/L0.9953.
Preferably, the HCR volume concentration is 11.5%.
The technical effect of adopting the technical scheme is as follows: the nucleic acid molecules in the HCR solution can completely protect the nano gold particles from being aggregated by salt ions, and the optimal effect is achieved.
Preferably, the concentration of the NaCl solution is 1 mol/L.
The technical effect of adopting the technical scheme is as follows: after the high-concentration salt is added into the nano-gold solution, the salt ions can destroy the surface charge balance of the nano-gold particles, so that the aggregation light absorption value of the nano-gold particles is transferred from 520nm to 620nm, and when the concentration of the NaCl solution is 1mol/L, the nano-gold particles are completely aggregated, so that the optimal effect is achieved.
Preferably, the incubation temperature is 37 ℃ and the incubation time is 30 min.
The technical effect of adopting the technical scheme is as follows: the salt ion aggregation nano-gold solution has the best effect when the nano-gold solution is incubated at 37 ℃ for 30 min.
The invention also provides application of the ampicillin residue detection method in detection of ampicillin residues in animal-derived food
The principle of the nano gold nucleic acid aptamer HCR amplification colorimetric method is as follows: as shown in fig. 1, the gold nanoparticles are metal particles, the surfaces of which have negative charges, and the gold nanoparticles are dispersed in a solution and maintained in a stable state due to the existence of mutual repulsive force between the particles. When the hairpin probe is added into the system, the single-chain sticky end of the probe and the surface of the nano-gold particle are adsorbed around the nano-gold through electrostatic interaction, and when high-concentration salt is added into the system, the nano-gold still exists in a dispersed mode and does not aggregate due to the protection of the hairpin probe. When Ampicillin (AMP) as a target is present in the system, AMP interacts with its aptamer AMP-1 to open the hairpin of AMP-1, a portion of the AMP-1 sequence is complementary to the single-stranded sticky end of hairpin probe H1, thus resulting in the opening of the H1 hairpin, while the end sequence of H1, due to the complementarity of the single-stranded end sequence of H2, results in the opening of the double-stranded hairpin of H2, and H2 sequence is designed with a sequence complementary to the end of the sticky single-stranded H1, thus H2 in turn results in the opening of a new H1 strand. Therefore, the two chains H1 and H2 are continuously hybridized circularly to form hybrid double-stranded DNA which extends infinitely, so that the amplification of signals is realized. As a double helix result of the double-stranded DNA, the double-stranded DNA shows electronegativity due to the exposure of a phosphate skeleton, and the electronegativity on the surface of the nanogold are mutually repelled, so that the nanogold particles are unprotected, and are aggregated at high salt concentration. Under the principle, the invention seeks an optimal method for detecting ampicillin by optimizing experimental conditions.
According to the technical scheme, compared with the prior art, the method for detecting ampicillin residues in animal-derived food disclosed by the invention has the following beneficial effects:
(1) the invention further initiates a cascade cross-complementary amplification effect by designing and developing a hairpin DNA probe, taking an aptamer as an initiation chain and initiating a complementary effect of the aptamer and the hairpin DNA probe through the combination of the aptamer and ampicillin, and finally realizes the method for efficiently detecting ampicillin.
(2) The invention designs and synthesizes a DNA hairpin of aptamer and two DNA hairpin probes, and realizes the cascade amplification of signals by utilizing a sequence complementary hybridization method between the hairpins. Research shows that the sensitivity range of the method is 0-1.4 mu mol/L, the lowest detection limit can reach 10nmol/L, the specificity is good, no non-specific reaction is caused to other types of antibiotics, and the recovery rate of fish meat and pork is 92.3% -103.27%.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of a nano-gold nucleic acid aptamer HCR amplification colorimetry.
FIG. 2 is a diagram showing the characteristics of the nanogold solution in example 1 of the invention.
Wherein, A is the absorption spectrum of the nano-gold solution, B is the absorption spectrum of the solution after the high-concentration salt is added, C is the color change of the nano-gold solution after NaCl with different concentrations is added, D is the state of the nano-gold solution under a transmission electron microscope, and E is the aggregation state of the nano-gold solution under the electron microscope after the high-concentration salt is added.
FIG. 3 is an optimization chart of experimental conditions in example 1 of the present invention.
Wherein, A is the optimization of NaCl concentration, and B is the optimization of HCR solution usage.
FIG. 4 is a graph showing the performance evaluation of the ampicillin detection system in example 1 of the present invention.
Wherein A is the sensitivity evaluation of the detection system on ampicillin, B is a standard curve for detecting ampicillin, and C is the specificity evaluation of the detection system.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Ampicillin, obtained from Sigma, sodium chloride, trisodium citrate, chloroauric acid (HAuCl)4) Are all analytically pure from Shanghai Chemicals, Inc.
Example 1
The embodiment provides a method for detecting ampicillin residues in animal-derived food, which specifically comprises the following steps:
s1, synthesis of ampicillin aptamer and matched hairpin DNA probe
The sequence of the aptamer for ampicillin is known from the prior art as:
5’-GCGGGCGGTTGTATAGCG-3’；SEQ ID NO.4，
the ampicillin aptamer sequence was redesigned on this basis as required by the HCR amplification technique as follows:
AMP-1：5’-CCGCCCGCTTAGTAGTTACGCTTACCTCTTGTTAGCGGG CGGTTGTATAGCGG-3’；SEQ ID NO.1；
the sequence of the hairpin DNA probe was:
H1：5’-CATCTCGGTTTGGCTTTCTTGTTACGGGCATTAGTAGTTACG CTTACC-3’；SEQ ID NO.2；
H2：5’-TAACAAGAAAGCCAAACCGAGATGGGTAAGCGTAACTAGT AATGCCCG-3’；SEQ ID NO.3；
all nucleic acid sequences were synthesized by Shanghai Bioengineering, Inc.
S2 preparation of hybrid chain melting (HCR) solution
Synthetic nucleic acid sequence, using ddH2O diluted to 1. mu.M of the nucleic acid working solution for use. And (3) respectively taking 100 mu L of the diluted nucleic acid solutions of H1, H2 and AMP from a PCR tube, cooling to 25 ℃ within 1H for 10min, then taking an EP tube, adding 100 mu L of the treated aptamer and the hairpin probe, and uniformly mixing to obtain a solution after HCR, and storing at-20 ℃ for later use.
S3 preparation of nano gold solution
The chloroauric acid is prepared into 200mL of a clear and transparent 0.01 percent chloroauric acid solution. The magnetic stirrer was turned on and heated with constant stirring. When boiling of the liquid in the beaker was observed, 5mL of a 1% solution of trisodium citrate was added. Boiling and stirring continuously, and finally turning the solution in the beaker from light gray to blue and purple to wine red, namely the nano-gold solution. Stopping heating, keeping stirring until the solution is cooled to room temperature, subpackaging and storing at 4 ℃.
Characterization of the above nanogold solution: the aggregation state of the nanogold solution was characterized using a transmission electron microscope. The experiment is divided into two groups, (a) 4mL of the prepared nano gold solution is taken; (b) and (3) taking 3mL of nanogold and 1mL of NaCl solution (4M), respectively dropwise adding the two solutions to the surface of the copper mesh according to requirements, and airing at room temperature for natural drying. The sample is shot by a JEM-1200EX transmission electron microscope, and the particle size of the Nano gold is analyzed by using Nano Measurer software on a computer.
S4 ampicillin colorimetric sensing method based on HCR nanogold detection
Adding 100 mu L of nano gold solution and optimized HCR solution into each melting tube, adding ampicillin with different concentrations into the test tube, incubating for 10min at room temperature, finally adding 100 mu L of NaCl into all the tubes, incubating at room temperature, detecting absorbance by an enzyme-labeling instrument, and analyzing to obtain a linear equation; and testing the absorbance of the sample to be tested by the same method, substituting the absorbance into a linear equation, and obtaining the residual amount of ampicillin in the sample.
In order to obtain the characterization of the nanogold, the invention also carries out a series of tests, which specifically comprise the following steps:
and respectively adding the newly prepared nano gold solution into the cuvette, and setting double distilled water as a blank control. Setting computer software parameters: the scanning wavelength is 400-800nm, and the scanning step length is 5 nm. The results showed that the newly prepared nanogold solution showed a wine red color, and the highest absorption peak appeared at 520nm, after which the absorbance gradually decreased with increasing wavelength (as shown in fig. 2A). 3mL of Nanogold +1mL of NaCl (1M, 2M, 4M) solution were mixed well and the color change was observed, and it was found that the solution gradually changed from wine red to blue as the salt concentration increased (as shown in FIG. 2C). The absorbance was measured by taking the solution to which 4M NaCl was added, and the highest absorption peak was found to shift to 620nm and there was only one absorption peak (as shown in FIG. 2B), so it was presumed that the nanogold solution had been completely accumulated with the salt at this time. The dispersion state and particle size of the particles of the nanogold solution and the aggregated solution were measured using an electron microscope, and the results showed that the newly prepared nanogold solution was in a dispersion state and particle size was about 13nm (as shown in fig. 2D), and that the nanogold particles aggregated together to exist in a mass form (as shown in fig. 2E) after the addition of 4M NaCl solution.
In order to obtain the optimal experimental conditions, the invention also optimizes the experimental conditions, and specifically comprises the following steps:
(1) optimizing NaCl concentration: NaCl solutions with different concentrations are respectively added into 100 mul of the nano gold solution, after incubation for 10min at room temperature, the light absorption value of each sample is measured by a multifunctional microplate reader, the optimal NaCL concentration is searched, and the results are shown in Table 1.
After the high-concentration salt is added into the nano-gold solution, the salt ions destroy the surface charge balance of the nano-gold particles to cause the aggregation light absorption value of the nano-gold particles to be transferred from 520nm to 620nm, so that after a concentration gradient NaCl solution is added into the nano-gold solution, the result shows that the ratio of the light absorption value A620/520 is gradually increased along with the increase of the NaCl concentration, and a peak value is reached when the concentration of the NaCl solution is 1mol/L, which indicates that the nano-gold particles are completely aggregated at this time, so that the optimal concentration of the NaCl solution is determined to be 1mol/L (as shown in FIG. 3A).
TABLE 1 NaCl concentration optimization data sheet
(2) Optimization of melting time: after mixing the nanogold solution with the HCR solution, the incubation time was set as a variable. The temperature at which the difference between the sample and blank groups was the greatest was taken as the optimal melting temperature.
A series of temperature gradients and optimization experiments of different reaction times show that the salt ion aggregation nano-gold solution has the best effect when the HCR solution and the nano-gold are incubated for 30min at 37 ℃, so the selected conditions are the optimal reaction conditions.
(3) Optimizing the use amount of the HCR solution: and (3) adding 100 mu l of nano gold solution into the sterilized centrifuge tube, and then respectively adding HCR solutions with different volumes. Subsequently, 100. mu.l of a 1M NaCl solution was added to the tube. After room-temperature incubation, detection and analysis are carried out by an enzyme-labeling instrument, and the method specifically comprises the following steps:
adding 100 μ l of nanogold solution, adding HCR solutions with different volumes respectively, incubating at 37 deg.C for 30min, adding 100 μ l of 1M NaCl solution into the tube, and detecting and analyzing the absorbance at 520nm and 620nm with microplate reader, the data are shown in Table 2. The results show that the reaction reached an equilibrium state when 30. mu.l of HCR solution was added (as shown in FIG. 3B), indicating that the nucleic acid molecules in the HCR solution were able to completely protect the gold nanoparticles from aggregation by salt ions, and therefore the final concentration of HCR solution, which is 30. mu.l of HCR solution, was selected to be 11.5% (v/v) as the optimum concentration.
Table 2 optimization data table of HCR solution usage
The invention also carries out performance evaluation on the ampicillin detection system, and specifically comprises the following contents:
(1) evaluation of System sensitivity
In order to find out the detection sensitivity of HCR amplification method, ampicillin is added into the optimized detection system with the concentration range of 0-2 mu mol/L for gradient dilution, wherein the specific concentration is 0 mu mol/L, 0.05 mu mol/L, 0.1 mu mol/L, 0.2 mu mol/L, 0.4 mu mol/L, 0.6 mu mol/L, 0.8 mu mol/L, 1 mu mol/L, 1.2 mu mol/L, 1.4 mu mol/L, 1.6 mu mol/L, 1.8 mu mol/L and 2 mu mol/L. The results are shown in the table, but it can be seen that the absorbance ratio of the system gradually increases with the increase of the ampicillin concentration, and the fitting equation is as follows: y is-0.0087 x2+0.261x-0.1307，R20.9924 (as shown in fig. 4A). Wherein when the concentration of ampicillin is in the range of 0-1.2 mu mol/L, the linear relation is shown, the linear equation is that y is 0.1636x +0.025, R20.9953 (as shown in fig. 4B), the lowest detection limit can reach 10nmol/L, and the above results show that the method has higher sensitivity.
TABLE 3 data table of HCR amplification method detection sensitivity
(2) Evaluation of System specificity
In order to verify the specificity of the detection system, antibiotics such as gentamicin, tetracycline, kanamycin and neostreptomycin are selected for specificity analysis with ampicillin, the final concentration of each antibiotic is 1 mu mol/L, the detection result shows that when the antibiotics are replaced by other antibiotics, the absorbance value of A620 of the reaction liquid is very low, and the absorbance value ratio of ampicillin is obviously higher than that of other antibiotics (as shown in figure 4C), which shows that the detection system only has specificity reaction on ampicillin.
The invention also detects the actual sample, which comprises the following contents:
in order to test the practical application capability of the detection method, two samples of fish and pork are selected and verified by a labeling recovery method. Extracting with acetonitrile, extracting with C18 column, adding ampicillin standard substances with different concentrations, and detecting with established experimental method. The results are shown in table 1, and the recovery rate of ampicillin is 92.3% -103.27%, and the experimental results show that the nano-gold aptamer HCR amplified colorimetric method has good detection performance.
TABLE 4 ampicillin assay results in animal foods
In conclusion, the method adopts the nano-gold nucleic acid aptamer hybridization chain reaction colorimetric method to detect the ampicillin, has the advantages of simple operation, quick reaction, low cost and the like, and is favorable for large-scale popularization. The invention designs and synthesizes a DNA hairpin of aptamer and two DNA hairpin probes, and realizes the cascade amplification of signals by utilizing a sequence complementary hybridization method between the hairpins. Research shows that the sensitivity range of the method is 0-1.4 mu mol/L, the lowest detection limit can reach 10nmol/L, the specificity is good, no non-specific reaction is caused to other types of antibiotics, and the recovery rate of fish meat and pork is 92.3% -103.27%. The method is convenient to operate, good in specificity and high in sensitivity, and is a novel method for detecting ampicillin residues.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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Claims (10)
1. A detection method of ampicillin residues is characterized in that firstly, an ampicillin aptamer sequence and a matched hairpin DNA probe are designed and prepared into HCR solution, and then the HCR amplified nano-gold colorimetric method is utilized to determine the ampicillin residues through the HCR solution and the nano-gold solution;
wherein the sequence of the ampicillin aptamer is as follows:
AMP-1：5’-CCGCCCGCTTAGTAGTTACGCTTACCTCTTGTTAGCGGGCGGTTGTATAGCGG-3’；SEQ ID NO.1；
the sequence of the hairpin DNA probe was:
H1：5’-CATCTCGGTTTGGCTTTCTTGTTACGGGCATTAGTAGTTACGCTTACC-3’；SEQ ID NO.2；
H2：5’-TAACAAGAAAGCCAAACCGAGATGGGTAAGCGTAACTAGTAATGCCCG-3’；SEQ ID NO.3。
2. the method for detecting ampicillin residue as defined in claim 1, comprising the steps of:
s1, synthesis of ampicillin aptamer and matched hairpin DNA probe: designing the sequence of the ampicillin aptamer as follows: AMP-1, with the sequence of the hairpin DNA probe: h1 and H2, all of which are synthesized by Shanghai Bioengineering, Inc.;
s2, preparation of HCR solution: the H1, H2 and AMP-1 nucleic acid sequences in step S1 were used with ddH2O are respectively prepared into 1 mu M of nucleic acid working solution and respectively melted at 90-95 ℃ by 1-20min, slowly cooling to 4-30 ℃ within 0.5-2H, uniformly mixing the treated AMP-1 aptamer with the treated H1 and H2 hairpin type nucleic acid probes to obtain HCR solution, and storing at-80 to-20 ℃ for later use;
s3, preparing a nano gold solution: preparing a clear and transparent 0.01-0.1% chloroauric acid solution from chloroauric acid, continuously heating to boil under stirring, adding a 0.1-5% trisodium citrate solution, continuously boiling and stirring, changing the solution from light gray to blue and purple in a beaker to finally obtain wine red, namely a nanogold solution, keeping stirring until the solution is cooled to room temperature, subpackaging, storing at 4-30 ℃, and representing the aggregation state of the nanogold solution by using a transmission electron microscope;
s4, detection of ampicillin residues: respectively taking the nanogold solution and the HCR solution, adding ampicillin with different concentrations, incubating for 0-120min at room temperature, adding 0.1-4M NaCl solution, incubating at room temperature, detecting absorbance by an enzyme-linked immunosorbent assay, and analyzing to obtain a linear equation; and testing the absorbance of the food to be tested by adopting the same method, substituting the absorbance into a linear equation, and obtaining the residual amount of ampicillin in the food.
3. The method of detecting ampicillin as claimed in claim 2, wherein in step S2, the volume ratio of the treated AMP-1 aptamer to the treated H1 and H2 hairpin nucleic acid probe is: (1): (1-10): (1-10).
4. The method of claim 2, wherein in step S3, the volume ratio of the chloroauric acid solution to the trisodium citrate solution is: (100-2000): (1-5).
5. The method for detecting ampicillin residue as claimed in claim 2, wherein in step S4, the volume ratio of said nanogold solution to said HCR solution to said sodium chloride solution is: (1): (1-10): (1-10).
6.The method of claim 2, wherein in step S4, the linear equation is y 0.1636x +0.025, R2The detection range for ampicillin was 0-1.2. mu. mol/L0.9953.
7. The method of detecting ampicillin as claimed in claim 2, wherein said HCR is at 11.5% by volume.
8. The method for detecting ampicillin as claimed in claim 2, wherein said NaCl solution has a concentration of 1 mol/L.
9. The method of detecting ampicillin residue as claimed in claim 2, wherein said incubation is carried out at 37 ℃ for 30 min.
10. Use of the method for detecting ampicillin residue as defined in any one of claims 1 to 9 for detecting ampicillin residue in a food product of animal origin.
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